Putative Androgen Response Elements Research Articles

Gonadal androgens are associated with decreased type I interferon production by plasmacytoid dendritic cells and increased IgG titres to BNT162b2 following co-vaccination with live attenuated influenza vaccine in adolescents.

mRNA vaccine technologies introduced following the SARS-CoV-2 pandemic have highlighted the need to better understand the interaction of adjuvants and the early innate immune response. Type I interferon (IFN-I) is an integral part of this early innate response that primes several components of the adaptive immune response. Women are widely reported to respond better than men to tri- and quadrivalent influenza vaccines. Plasmacytoid dendritic cells (pDCs) are the primary cell type responsible for IFN-I production, and female pDCs produce more IFN-I than male pDCs since the upstream pattern recognition receptor Toll-like receptor 7 (TLR7) is encoded by X chromosome and is biallelically expressed by up to 30% of female immune cells. Additionally, the TLR7 promoter contains several putative androgen response elements, and androgens have been reported to suppress pDC IFN-I in vitro. Unexpectedly, therefore, we recently observed that male adolescents mount stronger antibody responses to the Pfizer BNT162b2 mRNA vaccine than female adolescents after controlling for natural SARS-CoV-2 infection. We here examined pDC behaviour in this same cohort to determine the impact of IFN-I on anti-spike and anti-receptor-binding domain IgG titres to BNT162b2. Through flow cytometry and least absolute shrinkage and selection operator (LASSO) modelling, we determined that serum-free testosterone was associated with reduced pDC IFN-I, but contrary to the well-described immunosuppressive role for androgens, the most bioactive androgen dihydrotestosterone was associated with increased IgG titres to BNT162b2. Also unexpectedly, we observed that co-vaccination with live attenuated influenza vaccine boosted the magnitude of IgG responses to BNT162b2. Together, these data support a model where systemic IFN-I increases vaccine-mediated immune responses, yet for vaccines with intracellular stages, modulation of the local IFN-I response may alter antigen longevity and consequently improve vaccine-driven immunity.

P-476 Metformin ameliorated testosterone-induced ferroptosis via inhibiting ferritinophagy in trophoblasts of polycystic ovary syndrome (PCOS) placentas

Abstract Study question Does trophoblasts of PCOS patients with aberrant iron metabolism, and how does metformin ameliorate androgen-induced ferroptosis in trophoblasts in vivo and in vitro? Summary answer Metformin ameliorates placental development and attenuates spontaneous miscarriage rate in early gestation by inhibiting ferritinophagy in trophoblasts of PCOS patients with hyperandrogenism. What is known already PCOS is a common reproductive endocrine disease in women of reproductive age, and a large proportion of patients present with hyperandrogenism. Studies have found that the incidences of spontaneous miscarriage and adverse pregnancy outcomes in PCOS patients are higher than those in healthy controls, which placental dysfunction is an important factor. It is related to mitochondrial dysfunction, oxidative stress and metabolic disorders. Ferroptosis is an iron-dependent and phospholipid peroxidation-mediated cell death. Studies have shown that iron ions participate in a variety of biochemical reactions in humans, and its abnormal metabolism may lead to some diseases especially in the reproductive system. Study design, size, duration Villi at gestational weeks of 6-8 were collected from 18 healthy controls and 18 women with PCOS. Human trophoblast stem cells (hTSCs) were isolated from fresh villi (6-8 weeks, n = 3) and cultured with testosterone, ferroptosis or autophagy inhibitors, metformin, or NCOA4 small interfering RNA. A dihydrotestosterone (DHT)-induced PCOS C57BL/6 mouse model was treated with or without metformin (3 months before mating) (n = 20 per group). Mouse placentas were obtained at embryonic days 11.5 (n = 50 per group). Participants/materials, setting, methods The levels of iron ions and lipid peroxidation were measured by PGSK staining, BODIPYC11 staining and MDA assay kit. The expressions of ferroptosis-related genes and proteins were assessed by qPCR, western blot and immunofluorescence. The binding of androgen receptor (AR) and NCOA4 to the putative androgen response element (ARE) was examined by ChIP, EMSA and luciferase assay. The influence of metformin on ferritinophagy in placentas of PCOS mice was confirmed by western blot and immunofluorescence. Main results and the role of chance The levels of iron ions and lipid peroxidation in villi at gestational weeks of 6-8 were higher in PCOS patients than in healthy controls. In vitro experiments, testosterone-induced ferroptosis was ameliorated by ferroptosis inhibitors, and ferritinophagy was mitigated by autophagy inhibitors or NCOA4 knockdown in primary cultured hTSCs. Testosterone induced ARs into the nucleus, where the recruitment of AR homodimers along with NCOA4 to the positive ARE on the SQSTM1 promoter, leading to autophagy induction and degradation of ferritin heavy chain 1 (FTH1) in autophagosomes, resulting in the release of ferritin bound irons as free ferrous irons. Metformin ameliorated testosterone-induced ferroptosis in hTSCs to a certain extent by inhibiting ferritinophagy. Compared to those of control mice, the placentas of DHT-treated PCOS mice showed increased levels of iron ions and lipid peroxidation, and altered expressions of ferroptosis-related proteins. Hyperandrogenism also induced ferritinophagy in labyrinthine zone of placentas in PCOS mice. Metformin ameliorated placental development was associated with decreased iron ions accumulation by inhibiting ferritinophagy in PCOS mouse placentas, which observably attenuated embryo absorption rate and spontaneous miscarriage incidence in the early gestation of PCOS mice. Limitations, reasons for caution The numbers of PCOS patients and mouse placentas in this study were small. The placental labyrinthine zone was not separated from the endocrine junctional zone and maternal decidua for molecular analyses. In addition, it would be better to isolated and cultured mouse TSCs to explore mechanisms on ferritinophagy in further. Wider implications of the findings This study provides further evidence for metformin as a PCOS treatment, which ameliorates placental development and attenuates spontaneous miscarriage rate in early gestation. Trial registration number not applicable

Consulting prostate cancer cohort data uncovers transcriptional control: Regulation of the MARCH6 gene

Cholesterol accumulation is a hallmark of prostate cancer (PCa) enabled by the upregulation of its synthesis, which presents a potential therapeutic target. This pathway is suppressed by the E3 ubiquitin ligase membrane-associated RING-CH-type finger 6 (MARCH6); however, little is known of MARCH6 regulation, particularly at the transcriptional level. Here, we consulted large transcriptomic PCa datasets to investigate transcription factors and DNA sequence elements that regulate the MARCH6 gene. Amongst 498 primary PCa tissues of The Cancer Genome Atlas, we identified a striking positive correlation between MARCH6 and androgen receptor (AR) gene expression (r = 0.81, p < 1 × 10−117) that held in other primary tumour datasets. Two putative androgen response elements were identified in the MARCH6 gene using motif prediction and mining of publicly accessible chromatin immunoprecipitation-sequencing data. However, MARCH6 expression was not androgen-responsive in luciferase reporter and qRT-PCR assays. Instead, we established that the MARCH6–AR correlation in primary PCa is due to common regulation by the transcription factor Sp1. We located a region 100 bp downstream of the MARCH6 transcriptional start site that contains three Sp1 binding sites and strongly upregulates promoter activity. The functionality of this region, and Sp1-mediated upregulation of MARCH6, was confirmed using pharmacological and genetic inhibition of Sp1. Moreover, modulation of Sp1 activity affected the stability of squalene monooxygenase, a cholesterol biosynthesis enzyme and MARCH6 substrate. We thus establish Sp1 as the first known regulator of the MARCH6 gene and demonstrate that interrogation of transcriptomic datasets can assist in the de novo inference of transcriptional regulation.

Elevated androgen levels induce hyperinsulinemia through increase in Ins1 transcription in pancreatic beta cells in female rats.

Hyperandrogenism is associated with hyperinsulinemia and insulin resistance in adult females. We tested whether androgens dysregulate pancreatic beta cell function to induce hyperinsulinemia through transcriptional regulation of insulin gene (Ins) in the islets. Adult female Wistar rats implanted with dihydrotestosterone (DHT; 7.5-mg, 90-d release) or placebo pellets were examined after 10 weeks. DHT exposure increased plasma DHT levels by 2-fold similar to that in polycystic ovary syndrome in women. DHT exposure induced hyperinsulinemia with increased HOMA-IR index in fasting state and glucose intolerance and exaggerated insulin responses following glucose tolerance test. DHT females had no change in islet number, size and beta cell proliferation/apoptosis but exhibited significant mitochondrial dysfunction (higher ADP/ATP ratio, decreased mtDNA copy number, increased reactive oxygen production and downregulation of mitochondrial biogenesis) and enhanced glucose-stimulated insulin secretion. Ins expression was increased in DHT islets. In vitro incubation of control islets with DHT dose dependently stimulated Ins transcription. Analysis of Ins1 gene revealed a putative androgen responsive element in the promoter. Chromatin-immunoprecipitation assays showed that androgen receptors bind to this element in response to DHT stimulation. Furthermore, reporter assays showed that the promoter element is highly responsive to androgens. Insulin-stimulated glucose uptake in skeletal muscle was decreased with associated decrease in IRβ expression in DHT females. Our studies identified a novel androgen-mediated mechanism for the control of Ins expression via transcriptional regulation providing a molecular mechanism linking elevated androgens and hyperinsulemia. Decreased IRβ expression in the skeletal muscles may contribute, in part, to glucose intolerance in this model.

A teleost androgen promotes development of primary ovarian follicles in coho salmon and rapidly alters the ovarian transcriptome.

Recent studies using several teleost models have revealed that androgens increase the size of previtellogenic (primary and/or early secondary) ovarian follicles. To explore our hypothesis that androgens drive the development of primary follicles into early secondary follicles, and to determine the mechanisms underlying these androgenic effects, we exposed juvenile coho salmon to near-physiological and relatively sustained levels of the nonaromatizable androgen 11-ketotestosterone (11-KT). This resulted in significant growth of primary ovarian follicles after 10 and 20 days, with follicles after 20 days displaying a morphological phenotype characteristic of early secondary follicles (presence of cortical alveoli). Utilizing the same experimental approach, we then analyzed how 11-KT rapidly altered the ovarian transcriptome after 1 and 3 days of treatment. RNA-Seq analysis revealed that 69 (day 1) and 1,022 (day 3) contiguous sequences (contigs) were differentially expressed relative to controls. The differentially expressed contigs mapped to genes including those encoding proteins involved in gonadotropin, steroid hormone, and growth factor signaling, and in cell and ovarian development, including genes with putative androgen-response elements. Biological functions and canonical pathways identified as potentially altered by 11-KT include those involved in ovarian development, tissue differentiation and remodeling, and lipid metabolism. We conclude that androgens play a major role in stimulating primary ovarian follicle development and the transition into secondary growth.

PTTG1, A novel androgen responsive gene is required for androgen-induced prostate cancer cell growth and invasion

Androgens (AR) play an important role in initiation and progression of prostate cancer. It has been shown that AR exert their effects mainly through the androgen-activated AR which binds to androgen response elements (AREs) in the regulatory regions of target genes to regulate the transcription of androgen-responsive genes, thus, identification of AR downstream target gene is critical to understand androgen function in prostate cancer. In this study, our results showed that androgen treatment of LNCaP cells induced PTTG1 expression, which was blocked by the androgen receptor antagonist, Casodex. Bioinformatics analysis and experiments using PTTG1 promoter deletion mutants showed that the PTTG1 promoter contains a putative androgen response element (ARE), which localizes in the −851 to −836 region of the promoter. Androgen activated androgen receptor (AR) binding to this ARE was confirmed by Chromatin immunoprecipitation (ChIP) assay. Furthermore, Knockdown of PTTG1 expression using short hairpin RNA significantly reduced androgen-induced LNCaP cell growth and invasion. In addition, we showed PTTG1 is highly expressed in metastasis prostate cancer tissue. These results suggest that PTTG1 is a novel downstream target gene of androgen receptor and take part in prostate cancer proliferation and metastasis.

Prenatal testosterone exposure induces hypertension in adult females via androgen receptor-dependent protein kinase Cδ-mediated mechanism.

Prenatal exposure to excess testosterone induces hyperandrogenism in adult females and predisposes them to hypertension. We tested whether androgens induce hypertension through transcriptional regulation and signaling of protein kinase C (PKC) in the mesenteric arteries. Pregnant Sprague-Dawley rats were injected with vehicle or testosterone propionate (0.5 mg/kg per day from gestation days 15 to 19, SC) and their 6-month-old adult female offspring were examined. Plasma testosterone levels (0.84±0.04 versus 0.42±0.09 ng/mL) and blood pressures (111.6±1.3 versus 104.5±2.4 mm Hg) were significantly higher in prenatal testosterone-exposed rats compared with controls. This was accompanied with enhanced expression of PKCδ mRNA (1.5-fold) and protein (1.7-fold) in the mesenteric arteries of prenatal testosterone-exposed rats. In addition, mesenteric artery contractile responses to PKC activator, phorbol-12,13-dibutyrate, was significantly greater in prenatal testosterone-exposed rats. Treatment with androgen receptor antagonist flutamide (10 mg/kg, SC, BID for 10 days) significantly attenuated hypertension, PKCδ expression, and the exaggerated vasoconstriction in prenatal testosterone-exposed rats. In vitro exposure of testosterone to cultured mesenteric artery smooth muscle cells dose dependently upregulated PKCδ expression. Analysis of PKCδ gene revealed a putative androgen responsive element in the promoter upstream to the transcription start site and an enhancer element in intron-1. Chromatin immunoprecipitation assays showed that androgen receptors bind to these elements in response to testosterone stimulation. Furthermore, luciferase reporter assays showed that the enhancer element is highly responsive to androgens and treatment with flutamide reverses reporter activity. Our studies identified a novel androgen-mediated mechanism for the control of PKCδ expression via transcriptional regulation that controls vasoconstriction and blood pressure.

The presence of androgen receptor elements regulates ZEB1 expression in the absence of androgen receptor.

Zinc finger E-box binding homeobox 1 (ZEB1) is a transcription factor that plays a central role in the epithelial to mesenchymal transition (EMT) of cancer cell lines. Studies on its regulation have mostly focused on the negative 3'UTR binding of miR200c. Interestingly, it has been previously reported that androgen receptor (AR) regulates ZEB1 expression in breast and prostate cancers. In order to validate this, various ZEB1 promoter deletions were cloned into a luciferase reporter system to elucidate the contribution of two putative androgen response elements (AREs). The in vivo contribution of AR was also assessed in cell lines after R1881 treatment using qPCR with prostate specific antigen (PSA) as the positive control. We discovered that AR upregulates the levels of expression of ZEB1 10-fold on a luciferase promoter that only contains the distal ARE. However, when the proximal ARE is included, no additional activation is apparent with AR or its hormone independent variant, AR-V7. Furthermore, we demonstrate here that a promoter construct containing both AREs activates transcription of ZEB1 even in the AR-null cell lines DU145 and PC3. Incubation of the AR-positive cell line, LNCaP with R1881, failed to substantially increase the expression levels of ZEB1. Despite the presence of AREs in the promoter region, it appears that ZEB1 expression can be induced even without AR. In addition, the region around the distal ARE is a potent repressor in AR-null cell lines.

Prolactin/Stat5 and Androgen R1881 Coactivate Carboxypeptidase-D Gene in Breast Cancer Cells

Plasma membrane-bound carboxypeptidase-D (CPD) cleaves C-terminal arginine from extracellular substrates. In the cell, arginine is converted to nitric oxide (NO). We have reported that up-regulation of CPD mRNA/protein levels by 17β-estradiol and prolactin (PRL) in breast cancer cells, and by testosterone in prostate cancer cells, increased NO production and cell survival. The CPD promoter contains a consensus γ-interferon-activated sequence (GAS) and 3 putative androgen response elements (ARE.1, ARE.2, ARE.3) that could potentially bind PRL-activated transcription factor Stat5 (signal transducer and activator of transcription 5) and the liganded androgen receptor (AR), respectively. This study showed that synthetic androgen R1881 and PRL elevated CPD mRNA/protein levels in human MCF-7 and T47D breast cancer cells in a time-/dose-dependent manner. PRL/R1881-elevated CPD expression was blocked by actinomycin-D, and a CPD promoter construct containing these GAS and AREs was stimulated by PRL or R1881, indicating transcriptional regulation by both hormones. Luciferase reporter assays showed that GAS and the adjacent ARE.1 only were active. Mutation of GAS in the ΔGAS-CPD construct (ARE.1 intact) abolished CPD promoter activity in response to PRL and, surprisingly, to R1881 as well. ΔGAS-CPD promoter activity was restored by PRL+R1881 in combination, and enhanced by ectopic Stat5, but abolished by Stat5 gene knockdown. Chromatin immunoprecipitation analysis confirmed binding of activated Stat5 and liganded AR to GAS and ARE.1, respectively. Activated Stat5 also induced binding of unliganded AR to ARE.1, and liganded AR induced binding of unactivated Stat5 to GAS. In summary, PRL and R1881, acting through Stat5 and AR, act cooperatively to stimulate CPD gene transcription in breast cancer cells.

Testosterone regulates keratin 33B expression in rat penis growth through androgen receptor signaling.

Androgen therapy is the mainstay of treatment for the hypogonadotropic hypogonadal micropenis because it obviously enhances penis growth in prepubescent microphallic patients. However, the molecular mechanisms of androgen treatment leading to penis growth are still largely unknown. To clarify this well-known phenomenon, we successfully generated a castrated male Sprague Dawley rat model at puberty followed by testosterone administration. Interestingly, compared with the control group, testosterone treatment stimulated a dose-dependent increase of penis weight, length, and width in castrated rats accompanied with a dramatic recovery of the pathological changes of the penis. Mechanistically, testosterone administration substantially increased the expression of androgen receptor (AR) protein. Increased AR protein in the penis could subsequently initiate transcription of its target genes, including keratin 33B (Krt33b). Importantly, we demonstrated that KRT33B is generally expressed in the rat penis and that most KRT33B expression is cytoplasmic. Furthermore, AR could directly modulate its expression by binding to a putative androgen response element sequence of the Krt33b promoter. Overall, this study reveals a novel mechanism facilitating penis growth after testosterone treatment in precastrated prepubescent animals, in which androgen enhances the expression of AR protein as well as its target genes, such as Krt33b.

Androgen response element of the glycine N-methyltransferase gene is located in the coding region of its first exon

Androgen plays an important role in the pathogenesis of PCa (prostate cancer). Previously, we identified GNMT (glycine N-methyltransferase) as a tumour susceptibility gene and characterized its promoter region. Besides, its enzymatic product-sarcosine has been recognized as a marker for prognosis of PCa. The goals of this study were to determine whether GNMT is regulated by androgen and to map its AREs (androgen response elements). Real-time PCR analyses showed that R1881, a synthetic AR (androgen receptor) agonist induced GNMT expression in AR-positive LNCaP cells, but not in AR-negative DU145 cells. In silico prediction showed that there are four putative AREs in GNMT-ARE1, ARE2 and ARE3 are located in the intron 1 and ARE4 is in the intron 2. Consensus ARE motif deduced from published AREs was used to identify the fifth ARE-ARE5 in the coding region of exon 1. Luciferase reporter assay found that only ARE5 mediated the transcriptional activation of R1881. ARE3 overlaps with a YY1 [Yin and Yang 1 (motif (CaCCATGTT, +1118/+1126)] that was further confirmed by antibody supershift and ChIP (chromatin immunoprecipitation) assays. EMSA (electrophoretic mobility shift assay) and ChIP assay confirmed that AR interacts with ARE5 in vitro and in vivo. In summary, GNMT is an AR-targeted gene with its functional ARE located at +19/+33 of the first exon. These results are valuable for the study of the influence of androgen on the gene expression of GNMT especially in the pathogenesis of cancer.

Abstract 5455: Differential regulation of E2A (TCF3) by androgens in prostate cancer cells.

Abstract Introduction: E2A (TCF3) is a multifunctional basic helix loop helix (bHLH) transcription factor. E2A promotes cell differentiation, acts as a negative regulator of cell proliferation in normal cells and cancer cell lines and is required for normal B-cell development. Previous studies from our laboratory has shown that E2A expression is highly increased in prostate cancer as compared to normal prostate and that it acts as a tumor promoter in prostate cancer. Given the diverse biological pathways regulated/ influenced by E2A little is known about its regulation in prostate cancer. Experimental design: E2A expression in androgen sensitive LNCaP and insensitive C81 prostate cancer cell lines was determined by western blot after treatments with androgen receptor (AR) agonist R1881 and antagonist casodex. Putative Androgen Response Elements (ARE) were identified in the first intronic region of the E2A gene using some of the online bioinformatics tools and confirmed by Chromatin Immunoprecipitation (ChIP) with AR antibody and Luciferase reporter assays on the above mentioned cell lines after treatments with R1881 and casodex. Results: E2A expression was found to increase with the increasing aggrasiveness of prostate cancer cell lines as compared to normal prostate epithelial cell line RWPE1. When androgen responsive cell line LNCaP was treated with R1881 there was an increased expression of E2A which decreased upon treatment with antiandrogen casodex whereas the E2A expression remained unaltered upon similar treatments in androgen insensitive cell line C81. The first intronic region of the E2A gene was predicted to contain two putative ARE sites. ChIP after treatment of LNCaP and C81 cells with R1881 and casodex showed that the intronic region was bound by AR in LNCaP cells only in the presence of R1881, whereas C81 cells showed a pulldown with AR in presence as well as absence of R1881. Similar results were observed in luciferase reporter assays indicating that E2A is transactivated by AR in LNCaP cell lines whereas it is independent of androgens in C81 cell line. Furthermore, Luciferase reporter assays also confirmed that only one of the two predicted putative AREs was functionally active and responsible for the androgen mediated regulation of E2A. Conclusion: Our results indicate that E2A is differentially regulated in Prostate cancer cell lines. The increased expression of E2A and its role as a tumor promoter in prostate cancer cell lines may be contributed to its loss of androgen dependence as is evident in its progression from androgen dependent LNCaP to independent C81 cells. Acknowledgements: This study was supported by NIH/NCI RO1 CA128914 and NIH/NCRR/RCMI G12RR03062. Citation Format: Divya Patel, Jaideep Chaudhary. Differential regulation of E2A (TCF3) by androgens in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5455. doi:10.1158/1538-7445.AM2013-5455

Direct androgen regulation of PDE5 gene or the lack thereof

Inhibition of phosphodiesterase-5 (PDE5) is a well-known mechanism for the effective treatment of erectile dysfunction (ED). Androgen supplementation has also been prescribed for treating ED. However, it has been widely accepted that androgen can upregulate PDE5 expression, and thus creating a paradox in which a positive regulator of erectile function (androgen) could possibly increase the level of a negative regulator (PDE5). To solve this paradox, we conducted a systematic search of the PubMed and a non-systematic search of the Internet using PDE5, erectile, penis, testosterone and androgen as keywords. The retrieved papers were analyzed for data concerning the expression and regulation of PDE5 by androgens. Human and rat PDE5A gene sequences were retrieved from GenBank and computer-analyzed. The results showed that a putative androgen-response element (ARE) was reported in a study of human PDE5A gene promoter, and this prompted a separate study on whether androgen regulates PDE5 expression. The positive outcome in the latter study has since been cited in 17 review and editorial articles as the underlying mechanism for androgen's therapeutic effects on ED. In addition, five other research studies also reached the same conclusion. On the other hand, two independent studies on the genome-wide searches for androgen-regulated genes did not find PDE5A as a candidate. Sequence analysis conducted in this study also failed to find ARE in rat PDE5A gene. Two independent studies on Leydig cells also failed to find positive regulation of PDE5 expression by androgen. Two other studies found concomitant reduction of cavernous smooth muscle and PDE5 expression in castrated rats. One of these studies also found no effect of androgen on PDE5 expression in cultured cavernous smooth muscle cells. Thus, it appears that reduced PDE5 expression in castrated animals is due to reduced smooth muscle content and that PDE5A gene is not directly regulated by androgens.

Profiling of Androgen Response in Rainbow Trout Pubertal Testis: Relevance to Male Gonad Development and Spermatogenesis

The capacity of testicular somatic cells to promote and sustain germ cell differentiation is largely regulated by sexual steroids and notably androgens. In fish species the importance of androgens is emphasized by their ability to induce sex reversal of the developing fries and to trigger spermatogenesis. Here we studied the influence of androgens on testicular gene expression in trout testis using microarrays. Following treatment of immature males with physiological doses of testosterone or 11-ketotestosterone, 418 genes that exhibit changes in expression were identified. Interestingly, the activity of testosterone appeared stronger than that of 11-ketotestosterone. Expression profiles of responsive genes throughout testis development and in isolated germ cells confirmed androgens to mainly affect gene expression in somatic cells. Furthermore, specific clusters of genes that exhibit regulation coincidently with changes in the natural circulating levels of androgens during the reproductive cycle were highlighted, reinforcing the physiological significance of these data. Among somatic genes, a phylogenetic footprinting study identified putative androgen response elements within the proximal promoter regions of 42 potential direct androgen target genes. Finally, androgens were also found to alter the germ line towards meiotic expression profiles, supporting the hypothesis of a role for the somatic responsive genes in driving germ cell fate. This study significantly increases our understanding of molecular pathways regulated by androgens in vertebrates. The highly cyclic testicular development in trout together with functions associated with regulated genes reveal potential mechanisms for androgen actions in tubule formation, steroid production, germ cell development and sperm secretion.

Androgen pathway stimulates MicroRNA-216a transcription to suppress the tumor suppressor in lung cancer-1 gene in early hepatocarcinogenesis

Deregulation of microRNAs (miRNAs) is common in advanced human hepatocellular carcinoma (HCC); however, the ones involved in early carcinogenesis have not yet been investigated. By examining the expression of 22 HCC-related miRNAs between precancerous and cancerous liver tissues, we found miR-216a and miR-224 were significantly up-regulated, starting from the precancerous stage. Furthermore, the elevation of miR-216a was mainly identified in male patients. To study this gender difference, we demonstrated that pri-miR-216a is activated transcriptionally by the androgen pathway in a ligand-dependent manner and is further enhanced by the hepatitis B virus X protein. The transcription initiation site for pri-miR-216a was delineated, and one putative androgen-responsive element site was identified within its promoter region. Mutation of this site abolished the elevation of pri-miR-216a by the androgen pathway. One target of miR-216a was shown to be the tumor suppressor in lung cancer-1 gene (TSLC1) messenger RNA (mRNA) through the three target sites at its 3' untranslated region. Finally, the androgen receptor level increased in male liver tissues during hepatocarcinogenesis, starting from the precancerous stage, with a concomitant elevation of miR-216a but a decrease of TSLC1. The current study discovered the up-regulation of miRNA-216a by the androgen pathway and a subsequent suppression of TSLC1 as a new mechanism for the androgen pathway in early hepatocarcinogenesis.

Alanine Aminotransferase Regulation by Androgens in Non-hepatic Tissues

Alanine amino-transferases (ALTs) play a crucial role in drug development as a surrogate marker of liver injury where elevations in serum ALT activity are used to diagnose drug-induced liver damage. Two ALT isoforms have been characterized with disparate but overlapping tissue expression. ALT1 is primarily expressed in live; ALT2 is found in muscle and prostate tissues. We investigate ALT gene expression in diverse rodent tissues following administration of the steroidal androgen receptor (AR) agonist dihydrotestosterone and a novel tissue selective nonsteroidal agonist S-23. Putative AR regulation of ALT expression was determined in silico by an orthologous promoter androgen response element (ARE) search. Regulation was evaluated by transient transfection of ALT promoter region constructs and qRT-PCR experiments in cultured cells and in tissues following androgen administration. Several putative AREs were found in the proximal promoter regions of ALT1 and ALT2. AREs in ALT2 but not ALT1 were capable of AR-mediated transcription. ALT2 expression was affected by castration and androgen administration in muscle and prostate but not liver tissues. Androgen action in non-hepatic tissues, as opposed to xenobiotic toxicity alone, may contribute to increases in serum ALT activity following androgen administration.

Androgen regulated HN1 leads proteosomal degradation of androgen receptor (AR) and negatively influences AR mediated transactivation in prostate cells

We recently reported that hematological and neurological expressed 1 (HN1) is a ubiquitously expressed, EGF-regulated gene. Expression of HN1 in prostate cell lines down-regulates PI3K-dependent Akt activation. Here, we investigate whether HN1 is regulated by androgens through the putative androgen response elements (AREs) found in its promoter. Knockdown of HN1 expression by siRNA silencing leads to an increase in Akt(S473) phosphorylation, resulting in the translocation of androgen receptor (AR) to the nucleus; these effects can be abrogated by the non-specific Akt inhibitor LY294002 but not by the ERK inhibitor PD98059. Furthermore, HN1 overexpression correlates with an increase in ubiquitination-mediated degradation (a consequence of the decrease in S213/210 phosphorylation of AR), ultimately resulting in the down-regulation of AR-mediated expression of the KLK3, KLK4, NKX3.1 and STAMP2 genes. We also found that HN1 overexpression suppresses colony formation as well as R1881-mediated growth in LNCaP cells, while it has the opposite effect (increasing colony formation but not proliferation) in PC-3 and DU145 cells. Therefore, we suggest that HN1 maintains a balance between the androgen-regulated nuclear translocation of AR and steady-state Akt phosphorylation, predominantly in the absence of androgens. If so, the balance between cell growth and EGF- and AR-signaling must be tightly regulated by HN1. This work has important implications for prostate cancer research, as AR, EGFR and HN1 are known to be highly expressed in prostate adenocarcinomas.

The calcium sensor STIM1 is regulated by androgens in prostate stromal cells

Prostate development and maintenance in the adult results from an interaction of stromal and glandular components. Androgens can drive this process by direct action on the stroma. We investigated whether there was a direct link between androgens and another key regulator of stromal cells, intracellular Ca2+ ([Ca2+ ]i ). Prostate stromal cells were freshly obtained and cultures derived from patients with benign prostatic hyperplasia. Gene expression in dihydrotestosterone treated and untreated cells was compared using Affymetrix gene expression arrays and Ca2+ regulated features were identified by Gene Ontology (GO). Changes in [Ca2+]i were determined in Fluo-4 loaded cells. Androgen regulation was confirmed by chromatin immunoprecipitaion. Stromal cell cultures were sorted for expression of integrin α1 β1 , which enriched for cells expressing the androgen receptor (AR). We identified key functional categories, within the androgen-induced gene expression signature, focusing on genes involved in calcium signaling. From this analysis, stromal interaction molecule-1 (STIM1) was identified as a significantly differentially expressed gene with four relevant associated GO terms. DNA sequence analysis showed that the promoter region of STIM1 contained putative androgen response element sequences in which AR binding ability of STIM1 was confirmed. Androgens directly regulated STIM1 expression and STIM1 effects on store-operated calcium entry were inhibited by STIM1 knock-down. Reduced STIM1 expression in prostate stromal cells led to a reduction in basal Ca2+ levels, the amount of Ca2+ released by thapsigargin and a reduction in store filling following TG-induced store depletion. These results indicate that androgens modulate [Ca2+]i through the direct regulation of the STIM1 gene by AR binding to the STIM1 promoter.

Abstract 4984: Androgen response element of the glycine N-methyltransferase gene is located in first intron

Abstract BACKGROUND. The prostrate cancer (PCa) susceptibility gene glycine N-methyltransferase (GNMT) is associated with the metastasis potential of PCa cells. Androgens, which participate in transcriptional regulation via an androgen receptor (AR), play an important role in PCa development. Our two goals were to determine whether GNMT is regulated by androgen, and to map androgen response elements (AREs). METHODS. Real-time PCR was used to evaluate GNMT mRNA expression levels after LNCaP and DU145 cells were treated with R1881 (a synthetic AR agonist). Putative AREs were identified and tested using the MatInspector program and a luciferase reporter assay respectively. Electrophoretic mobility shift assay and chromatin immunoprecipitation were used to verify the binding properties of the GNMT-ARE motif. RESULTS. Results from real-time PCR analyses indicate that R1881 is capable of inducing significant GNMT mRNA levels in AR-positive LNCaP cells, but not in AR-negative DU145 cells. According to in-silico screening prediction data, putative ARE1, ARE2 and ARE3 are located within GNMT intron 1, and ARE4 is located within intron 2. According to results from a luciferase reporter assay, only ARE3 mediated the transcriptional activation effects of R1881. Results from an electrophoretic mobility shift assay show that AR binds to ARE3 in vitro. Chromatin immunoprecipitation assay results indicate AR interaction with the ARE3-containing region in vivo. CONCLUSION. Our data show that GNMT is an AR target gene, with its functional ARE located at +1114/+1128 in GNMT intron 1. We believe this information is valuable for clarifying the relationship between androgen and GNMT gene expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4984.

Androgen-regulated proteins in the rat ventral prostate: Androgen-regulierte Proteine der ventralen Rattenprostata

The rat ventral prostate synthetizes and secretes a number of relatively abundant proteins, such as Prostatic Binding Protein (PBP), the Proline-Rich Polypeptides (PRP), the 20 or 22-kDa protein and a prostatic protease, which respectively constitute approx. 50, 5, 10 and 2% of the secreted protein. The synthesis of these proteins is androgen-regulated and this is also the case for the concentration of the corresponding mRNAs. The existence of a putative androgen response element in the first intron of the PBP-C3 gene supports the possibility of a direct control of its transcription by androgen receptor complexes, but there is also evidence for the requirement of protein synthesis for mRNA stimulation. Finally, changes of tissue composition and cell-cell interactions between epithelial and stromal cells most probably play an important role in the global effect of androgens on the rat ventral prostate.